Team:HUST-China:Modeling

Modeling on Ecosystem Level
The “wake-up” problem
The permeation problem
Robustness Analysis

Interlab Study

SectionⅠ: Introduction

This year, iGEM invited and encouraged all teams to participate in the Second International InterLab Measurement Study in synthetic biology. We were required to measure the expression level of GFP when using three different promoters. Although It was the first time for our team to take part in Interlab Study, we successfully conducted the experiment and obtained the fluorescence data. Now, we are hoping that we can make contributions to Interlab Study.

Section Ⅱ: Provenance and Release

Individuals responsible for conducting InterLab study:

Create the devices: Zhi Zeng Conduct measurements: Guozhao Wu, Shuyan Tang and Zhi Zeng Process data: Shuyan Tang and Yee Zhan

Date of InterLab Study:

The measurement was obtained on August 27, 2015.

Did your team participate in the Extra Credit?

No. (Details can be found here: https://2015.igem.org/Tracks/Measurement/Interlab_study)

Do all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study?

Yes.

Section Ⅲ: Equipment Information

What type of incubator did you use to grow your cells?

THZ-D desktop constant temperature oscillator

If known, what was your incubator's throw (shaking diameter)?

Oscillation frequency: 20~350rpm

Amplitude: 26mm

Maximum capacity(each layer): 1000mlx6 or 500mlx9 or 250mlx12

Standard configuration: Spring rack

Dimension of the tray (mm): 400x340

Time range: 0~999minutes

Control temperature: (Environment temperature)Room temp+5℃~60℃

Temperature increment: 0.1℃

Inner temperature error: ±0.5℃

Display: LED

Input power: 350w

Size (mm): 650x500x480

What piece of equipment did you use to measure the devices?

FlexStation 3 Multi-Mode Microplate Reader

When was this equipment last calibrated?

August 1st, 2015

Who calibrated the equipment?

Tengteng Gong

What was the wavelength of light you used to excite the cells?

485nm

What was the filter/channel you used to capture the light emission from the cells?

Monochromators, tunable in 1.0 nm increments Wavelength range 400–750 nm

What was the sampling frequency?

50 times per second

Section Ⅳ: Protocol

Did you fill in the InterLab Protocol provided by the Measurement committee?

Yes (The InterLab Protocol is provided here: https://2015.igem.org/Tracks/Measurement/InterLab_Protocol)

Did you follow the InterLab Protocol provided by the Measurement committee?

Yes

How did you determine the final dataset that you are reporting?

Since theoretically the emission wavelength of GFP is up to 518nm and the test data showed 518nm got the peak signal, we chose Em518 data. We also tested LB medium as blank and the data was minus the blank.

Section Ⅴ: Measurement results for Interlab Study

Units Reported:

RFUs

We measured the three specific devices required by Interlab Study, using E.coli DH5α as our chassis.

1.Device 1: BBa_J23101 + BBa_I13504 (B0034-E0040-B0015) in the pSB1C3 backbone

2.Device 2: BBa_J23106 + BBa_I13504 (B0034-E0040-B0015) in the pSB1C3 backbone

3.Device 3: BBa_J23117 + BBa_I13504 (B0034-E0040-B0015) in the pSB1C3 backbone

We diluted each of our samples to an OD600 of 0.5 and we started measuring fluorescence when OD600 was within 5% of 0.5. We measured each sample in biological triplicates.

We obtained the fluorescence data by using 485nm as the excitation wavelength and 518nm as the emission wavelength. Measurement data were collected as relative fluorescence units.

Final fluorescence data were collected on August 27, 2015.

We divided fluorescence by relevant OD600 and used the results to draw the graph above. According to the graph, all the samples seemed to be normal except Device3: J23117+I13504. Sadly Device3 didn’t express GFP while Device1 had the highest fluorescence intensity, revealing that J23101 is a relative high strength promoter compared with J23106 and J23117.

The fluorescence results that we submitted to Interlab Study are below:

All the data below has been minus our blank control, LB medium.

	The First Replicate	The Second Replicate	The Third Replicate
Positive Control: BBa_I20270	1346.963	1640.876	1265.491
Negative Control:BBa_K950001	-58.27	-63.088	2.847

Sample	Biological Replicate 1	Biological Replicate 2	Biological Replicate 3	Mean	Standard Deviation
Device 1: BBa_J23101+ BBa_I13504	4737.16	4864.736	4715.018	4772.305	120.498
Device 2: BBa_J23106+ BBa_I13504	1271.075	860.502	1581.901	1237.826	299.201
Device 3: BBa_J23117+ BBa_I13504	-31.803	-28.755	51.543	-3.005	50.803